Development of process optimization for production of the recombinant modified vaccinia Ankara (rMVA) virus booster component of a novel multiprotein-expressing DNA/rMVA HIV vaccine candidate is proposed. In preclinical trials in macaques, DNA priming and rMVA boosting with Gag-Pol-Env-expressing immunogen for a chimera of simian and human immunodeficiency viruses (SHIV) has successfully controlled a highly pathogenic SHIV-89.6p challenge administered by the mucosal route at 7 months after the last immunization (Science 292:69). Clade B Gag-Pol-Env DNAs and rMVAs have been constructed in the laboratories of Dr. Harriet Robinson (Emory) and Dr. Bernard Moss (NIAID). These will be entering phase I trials sponsored by the NIAID HIV Vaccine Trials Network in early 2002. A major problem in preparing reagents for phase I trials was GMP production of the rMVA. Given the success of the multiprotein DNA/rMVA vaccine in preclinical models, the Emory Vaccine Center and Emory University helped start GeoVax, Inc., a company whose initial goal is the translation of the DNA/rMVA HIV-1 vaccine for human use. The overall goal of this proposal is to undertake the process engineering for GMP production of future lots of rMVA such that the vaccine used in phase III trials will have been produced under conditions that will support cost effective production of a licensed vaccine. The clade B Gag-Pol-Env rMVA grows well under standard laboratory conditions to titers as high as lxl0E10 pfu per 850 cm2 roller bottle. During GMP production for phase I trials, the best titers achieved were about 3x10E9 pfu per 850 cm2 roller bottle. The primary goal of this proposal is to achieve scale-up conditions that produce titers of at least lxl0E10 pfu and hopefully lxl0E11 pfu of rMVA per 850 cm2 roller bottle equivalent. The rMVA grows best on primary chick embryo fibroblast cells (CEF). Development of conditions for efficient large-scale production will use a novel scale-up methodology for CEF growth and virus production coupled with a logical design for growth optimization. CEF-derived vaccines have a long history for human use with a number of products approved and millions of doses administered. Our goal is to increase rMVA titers to lxl0E10 - lxl0E11 per 850 cm2 roller bottle equivalent to provide a safe and cost effective product to meet the global need for an HIV-1 vaccine.